Identification of 5-methyl-C in nucleic acid templates

• US 9,175,348 B2
• Filed: 04/23/2013
• Issued: 11/03/2015
• Est. Priority Date: 04/24/2012
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA), University of Chicago
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Korlach, Jonas, He, Chuan, Clark, Tyson A., Zhang, Liang, Lu, Xingyu
• Primary Examiner(s): Lu, Frank
• Application Number:
  US13/868,661
• Publication Number:
  US 20140004511A1
• Time in Patent Office: 924 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/91.51, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2523/115 oxidising agents
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases

Abstract

A method for identifying a 5-MeC in a template nucleic is provided. The method comprises providing a template having 5-MeC, converting the 5-MeC into a further modification selected from 5-caC and 5-FC. The converted template is then sequenced, and a change in sequencing is detected that is indicative of the further modification, allowing for identifying the 5-MeC in the template nucleic acid.

Claims

1. A method for identifying a 5-methylcytosine (5-MeC) in a template nucleic acid, the method comprising:

a) providing a template nucleic acid comprising the 5-MeC;

b) converting the 5-MeC into a modified base by treating the template nucleic acid with a Tet protein, wherein the modified base is selected from 5-carboxylcytosine (5-caC) and 5-formylcytosine (5-fC), thereby generating a modified template nucleic acid;

c) sequencing the modified template nucleic acid in the presence of fluorescently labeled nucleotides in a single-molecule sequencing reaction;

d) monitoring said single-molecule sequencing reaction, wherein said monitoring comprises measuring interpulse duration, which is the length of time between fluorescent pulses, and pulse width, which is the width of fluorescent pulses, during said single-molecule sequencing reaction, and the fluorescent pulses are generated during incorporation of single bases of said fluorescently labeled nucleotides into a nascent nucleic acid strand synthesized during said single molecule sequencing reaction; and

e) identifying the 5-methylcytosine (5-MeC) in the template nucleic acid by detecting a kinetic change during said sequencing the modified template nucleic acid, wherein said kinetic change is a kinetic change relative to a control template nucleic acid and comprises at least one change selected from the group consisting of a change in the interpulse duration and a change in the pulse width, wherein the presence of said kinetic change indicates the presence of the 5-MeC in the template nucleic acid.

2. The method of claim 1, wherein the template nucleic acid is a circular nucleic acid.

3. The method of claim 2, wherein said single-molecule sequencing reaction comprises rolling-circle synthesis of said nascent nucleic acid strand.

4. The method of claim 1, wherein the template nucleic acid is an RNA or DNA molecule.

5. The method of claim 1, wherein said single-molecule sequencing reaction comprises a polymerase.

6. The method of claim 1, wherein said monitoring said single-molecule sequencing reaction detects incorporation of said single bases of said fluorescently labeled nucleotides into the nascent nucleic acid strand and generates a sequence read.

7. The method of claim 6, wherein the kinetic change occurs at the modified base.

8. The method of claim 6, wherein the kinetic change occurs at one or more positions upstream or downstream of the modified base.

9. The method of claim 6, wherein said fluorescently labeled nucleotides are differentially labeled to be distinguishable from one another during said monitoring of said single-molecule sequencing reaction.

10. The method of claim 9, wherein each of said fluorescently labeled nucleotides comprises a fluorescent label linked to a phosphate group and the phosphate group is removed during said incorporation.

11. The method of claim 6, further comprising mapping the 5-MeC within the template nucleic acid, the mapping step comprising:

f) analyzing a portion of the sequence read to determine a sequence complementary to the template nucleic acid;

g) determining a sequence of the complement of the sequence complementary to the template nucleic acid; and

h) mapping the 5-MeC at a position in the template nucleic acid.

12. The method of claim 1, wherein the template nucleic acid further comprises a glucose-modified 5-hydroxymethylcytosine (5-hmC).

13. The method of claim 12, further comprising identifying the glucose-modified 5-hmC in the template nucleic acid by detecting a further kinetic change in said single-molecule sequencing reaction.